Protein kinase C epsilon and genetic networks in osteosarcoma metastasis.

Osteosarcoma (OS) is the most common primary malignant tumor of the bone, and pulmonary metastasis is the most frequent cause of OS mortality. The aim of this study was to discover and characterize genetic networks differentially expressed in metastatic OS. Expression profiling of OS tumors, and subsequent supervised network analysis, was performed to discover genetic networks differentially activated or organized in metastatic OS compared to localized OS. Broad trends among the profiles of metastatic tumors include aberrant activity of intracellular organization and translation networks, as well as disorganization of metabolic networks. The differentially activated PRKCε-RASGRP3-GNB2 network, which interacts with the disorganized DLG2 hub, was also found to be differentially expressed among OS cell lines with differing metastatic capacity in xenograft models. PRKCε transcript was more abundant in some metastatic OS tumors; however the difference was not significant overall. In functional studies, PRKCε was not found to be involved in migration of M132 OS cells, but its protein expression was induced in M112 OS cells following IGF-1 stimulation.

Characterization of the 12q15 MDM2 and 12q13-14 CDK4 amplicons and clinical correlations in osteosarcoma.

The chromosomal region 12q13-15 is recurrently amplified in osteosarcoma (OS), but its importance in bone tumor development remains unknown. Although there are two major candidate genes (MDM2, a TP53 downregulator, and CDK4, involved in cell cycle progression) considered to be the driving genes in this region, the size of the amplicon and number of genes involved have not been determined. In this study, we used 130 classical OS and 15 parosteal OS to determine MDM2 and CDK4 amplification frequency in OS. Tumors in which these genes were amplified were used to map the 12q13-15 amplified region and to determine its correlation with clinical prognosis. The 12q13-15 amplification was more prevalent in parosteal OS (67% of cases) than in high-grade classical OS (12%). Quantitative real-time PCR of MDM2, CDK4, and 25 other genes showed that this region contains two different amplicons: one at 12q15 centered on MDM2 and one at 12q13-14 centered on CDK4. Both regions were frequently co-amplified in both types of OS, and MDM2 and CDK4 amplification was correlated with higher expression levels for both genes. Univariate and multivariate analyses of clinical data indicated that classical OS patients whose tumors exhibited MDM2 amplification were more likely to be older at diagnosis (median age 32.6 vs. 17.8 years) and female (66.7 vs. 33.3%) than those without gene amplification. There was no association with other clinical parameters. In conclusion, co-amplification of MDM2 and CDK4 in two separate amplicons occurs frequently in parosteal OS and less so in classical high-grade OS.

Identification of rare variants in the hLIMD1 gene in breast cancer.

The hLIMD1 gene is located at chromosome 3p21 and was identified as a putative tumor suppressor gene using an elimination test assay. Chromosome 3p21 loci are frequently deleted in a number of cancers, including breast. The 3p21.3 locus harbors a number of tumor suppressor candidates, including LIMD1, a member of the ZYXIN family of genes. LIMD1 directly interacts with RB and is thought to play a role in suppressing tumor growth. To investigate whether mutations in the LIMD1 gene could potentially be involved in breast cancer, we used single-stranded conformation polymorphism analysis on DNA from 235 breast cancers and 95 controls. We identified four novel coding region alterations, including two amino acid substitutions at positions 255 and 302. The two remaining novel variants were found at amino acid positions 246 and 647 and encoded silent alterations. The rare Ser255Arg variant was identified in only sporadic breast tumors (2/165 tumors). Some ZYXIN proteins are phosphorylated by serine/threonine kinases, and the Ser255Arg change is located in a region phosphorylated on serine residues. Together, the data suggest that this variant may warrant further characterization.

COPS3 amplification and clinical outcome in osteosarcoma.

BACKGROUND: Amplification of several genes that map to a region of chromosome 17p11.2, including COPS3, was observed in high-grade osteosarcoma. These genes were also shown to be overexpressed and may be involved in osteosarcoma tumorigenesis. COPS3 encodes a subunit of the COP9 signalosome implicated in the ubiquitination and ultimately degradation of the P53 tumor suppressor. To determine the relation between COPS3 amplification, P53 mutation, and patient outcome in osteosarcoma, tumors from a large cohort of patients with high-grade osteosarcoma and long-term clinical follow-up were examined. METHODS: Quantitative real-time polymerase chain reaction (PCR) was performed to detect copy number changes for COPS3, as well as additional genes (NCOR1, TOM1L2, and PMP22) from the 17p11.2 amplicon, in 155 osteosarcomas from a prospective collection of tumors with corresponding clinical data. Univariate and multivariate analyses were performed to assess differences in survival between groups. RESULTS: Amplification of COPS3, detected in 31% of the osteosarcomas, was strongly associated with large tumor size (P=.0009), but was not associated with age at diagnosis, site, sex, and tumor necrosis. COPS3 amplification was significantly correlated with a shorter time to metastasis with an estimated hazard ratio (HR) of 1.61 (95% confidence interval [CI], 1.02-2.55) in univariate analysis (log-rank test, P=.042). However, in an a priori multivariate Cox model including the other clinical parameters, the HR for COPS3 amplification decreased to 1.32 (95% CI, 0.82-2.13, P=.25), mainly due to the strong correlation with tumor size. COPS3 amplification and P53 mutation frequently occurred in the same tumors, suggesting that these are not mutually exclusive events in osteosarcoma. Although not statistically significant, patients whose tumors exhibited both molecular alterations tended to be more likely to develop metastasis compared with patients with either COPS3 amplification or P53 mutation alone. CONCLUSIONS: COPS3 is the likely target of the 17p11.2 amplicon. COPS3 may function as an oncogene in osteosarcoma, and an increased copy number may lead to an unfavorable prognosis.